Journal of Plastic, Reconstructive & Aesthetic Surgery (2012) 65, e80ee86

The reinnervation pattern of wounds and scars aftertreatment with transforming growth factorb isoforms*

James Henderson a,*, Mark W.J. Ferguson b, Giorgio Terenghi c

aDepartment of Plastic and Reconstructive Surgery, Norfolk and Norwich University Hospital, NHS Trust,Norwich NR4 7UJ, UKbRenovo Ltd, Manchester, UKcBlond McIndoe Laboratories, Plastic and Reconstructive Surgery Research, University of Manchester, Manchester, UK

Received 8 May 2011; accepted 20 December 2011

KEYWORDSTGF beta;Transforming growthfactor;Wound reinnervation;Scar innervation

* This work was presented as part* Corresponding author. Tel.: þ44 0E-mail address: jh@jameshenderso

1748-6815/$-seefrontmatterª2011Bridoi:10.1016/j.bjps.2011.12.013

Summary Background: Wounds deprived of innervation fail to heal normally, and hypertro-phic scars may be abnormally innervated. Manipulation of wounds alters the subsequentdegree of scarring, and isoforms of transforming growth factor beta (TGFb) are well estab-lished in this role, whilst TGFb3 is undergoing clinical trials as an antiscarring agent for clinicaluse. It is unclear if treated wounds show changes in their innervation patterns as they matureinto scars.Methods: Mice underwent 1cm2 full thickness skin excisions which were treated with TGFb1 orTGFb3. Wounds were harvested between 5 and 84 days (nZ 6 at each time point). Sectionsunderwent histological scar assessment and immunohistochemical staining for protein geneproduct 9.5 (PGP9.5), a pan-neuronal marker, and the sensory neuropeptides calcitonin generelated peptide (CGRP) and substance P (SP).Results: There was no difference in the reinnervation pattern between the peripheral andcentral parts of the wounds.

Wounds treated with TGFb3 healed with dermal collagen organised more like normal skin,whereas TGFb1 treated wounds had abnormally orientated collagen within the scar comparedto control treated wounds. Nerve fibre growth into the wounds followed a similar pattern incontrol and treated wounds, with only one significant difference during the healing process-at 42 days, the density of nerve fibres immunostained with PGP9.5 within the scar was greaterthan in control wounds. By 84 days, the density of PGP9.5, CGRP and SP immunopositive fibreswere similar in control wounds and those treated with TGFb isoforms.

of JHs thesis for the degree of doctor of Medicine (MD).1603286286.n.net (J. Henderson).

tishAssociationofPlastic,ReconstructiveandAestheticSurgeons.PublishedbyElsevierLtd.All rightsreserved.

The reinnervation pattern of wounds and scars after treatment e81

Conclusions: Changes in reinnervation patterns of wounds treated with TGFb isoforms wereonly slightly different from those of control wounds, and by 84 days, the patterns were similar.ª 2011 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published byElsevier Ltd. All rights reserved.

Introduction

Cutaneous scarring is the macroscopic disturbance of thenormal structure and function of the skin as a consequence ofwound healing, and owing to changes in epidermal, dermal,and subcutaneous tissue at the time of wounding.1 Scarsgenerally exhibit abnormal sensation, often being insensate,but sometimespruritic,painful orhypersensitive.2Tissues failto heal normally after injury if their nerve supply is compro-mised, either forming ulcers3 or unstable scars4 and havea decreased inflammatory response.4,5 Denervated skin flapsin rats suffer greater tissue loss than innervated controls.6

Nerves within hypertrophic scars are more numerous7 aswell as thicker and more tortuous than controls.8

Elevated substance P (SP) levels have been found inpainful and pruritic hypertrophic human scars,9 whilecalcitonin gene related peptide (CGRP) levels wereincreased in the adjacent dermis. Both SP and CGRPimmunopositive axons have been found in the epidermis ofpainful hypertrophic scars and it was suggested that SPantagonism might reduce scar hypertrophy.10 Substance Plevels measured by ELISA are higher in hypertrophic burnscars than control scars or skin.11

Mouse excisional wounds have significantly elevatedlevels of CGRP immunopositive fibres growing into themduring healing, which reduce by 84 days to levels similar tocontrols. SP immunopositive nerve fibre density was foundto be persistently elevated in such scars.12

Three isoforms of TGFb are found in mammals. TGFb1 &TGFb2 cause deposition of fibronectin, fibrin and collagenand may worsen scarring.13 TGFb3 administration reducesscarring in animals14,15 and man.16 TGFb isoforms arestructurally similar to nerve growth factor and havefurthermore been found to increase NGF levels in ratneonatal brain injury17 and in peripheral nerves.18 Theeffects of exogenous TGFb isoforms on nerve fibre growthduring cutaneous wound healing are unknown.

TGFb1 is the most prevalent of the three mammalianisoforms, found in basal keratinocytes and in platelets.19

TGFb3 is only found at low levels in unwounded mouseskin.20

TGFb isoforms became of interest when higher levels ofTGFb3 and lower levels of TGFb1 and TGFb2 were found innon-scarring fetal wounds than adult wounds that healedwith scars.21 Exogenous addition of TGFb3 to adult woundsimproves scarring, with a decrease in the number ofinflammatory cells, and restoration of the neodermis toa form more like that of uninjured skin, but an increase inangiogenesis is seen following treatment of wounds withTGFb3.14 Transfecting wounds with additional TGFß1 mRNAled to increased type I collagen and decreased collagenasemRNA expression.22 TGFß1 has been implicated in keloidand hypertrophic scarring.23

Given the evidence that sensory innervation and woundhealing are so closely linked, it might be that treatmentsthat alter the scarring process also affect wound reinner-vation. The aim of this study was to investigate the growthof nerve fibres into healing wounds and scars, after theapplication of TGFb1 or TGFb3, both growth factors beingknown to alter the scarring process.

Materials and methods

All experiments were carried out in accordance with the UKanimal act (1986) and with appropriate legal and ethicalapproval. Male CD1 mice aged 8e10 weeks were individu-ally housed before and after the creation of a standardisedwound. Mice were anaesthetised with halothane, nitrousoxide and oxygen. Two 1 cm2 excisional wounds werecreated on the backs of 42 mice. The skin and panniculuscarnosum were removed and intradermal injections of200 ng TGFb1 or 200ng of TGFb3, were perfomed at themargins of one wound. The TGFb was diluted in100 ml ofbuffer in each case, and 25 ml was injected into each of thefour wound margins. Contralateral control wounds weresimilarly injected with 100 ml of phosphate buffered saline.Wounds were left to heal by granulation and no dressingwas applied. Twenty-four hours after creation of thewound, mice were anaesthetised again, and an identicalsecond dose of drug or control saline was administered.

Mice were killed by overdose of anaesthetic agent at 7,14, 21, 42 and 84 days (nZ 6 at each time point) and thewounds harvested after photographs were taken, allowingsubsequent determination of the area of the wound or scar.Determination of control wound areas was also made at fivedays postoperatively.

Harvesting, processing and analysis of the wound inner-vation was performed as previously described.12 Primaryantibodies were either rabbit anti human PGP9.5 (Affiniti,Exeter, UK dil 1/500), rabbit anti rat CGRP (Affiniti, dil1/3000) or rabbit anti cow SP antibodies (Affiniti, dil 1/5000). A fluorescein conjugated polyclonal goat anti-rabbitsecondary antibody was used (Vector Laboratories, Burlin-game, CA, USA dil 1/100).

Massons trichrome staining was carried out on separate15 mm sections of all wounds, allowing correlation of thewound width and architecture with the immunohisto-chemical findings. Massons stain was performed on six 7 mmsections from each day 84 scar (36 sections per treatmentgroup), and these sections were used to assess the degreeof organisation of collagen (severity of scarring). Twoseparate observers were blinded to the treatment thateach slide had received, and assessed the degree of orga-nisation of the collagen against untreated control woundsections, using a visual analogue scale scoring system whichhas been previously described.24

Figure 2 Massons stained 7 mm sections through unwoundedskin and 84-day-old scars from each of the treatment groups.Higher power views are shown in the right hand panels. Thecollagen fibre pattern in the TGFb3 treated wound most closelyresembles the “basket weave” appearance of the unwoundedskin; whilst the collagen fibres of the TGFb1 treated scar showsthe most densely packed fibres. Scale bars are 50 mm.

e82 J. Henderson et al.

Statistical analysis (Dunnetts C test) was performedusing SPSS software (SPSS, Chicago, IL) to compare themean staining densities of the imaged areas for each timepoint, allowing for comparison of measurements from theperipheral and central parts of wounds. Wound areas andscar severity scores on the visual analogue scale weresimilarly analysed.

Results

All the wounds epithelialised without complication by 14days and by 84 days the wound has become a mature scar.Wounds underwent contraction over the first week, reach-ing their final size by 7 days in all cases. The final woundarea was about a tenth of the size of the initial wounds(Figure 1).

The scar-altering treatments changed the degree ofcollagen organisation in the scars at 84 days as seen withMassons staining. Wounds treated with TGFb1 showedconsistently less organised collagen within the scarscompared to controls, whilst wounds treated with TGFb3showed collagen orientated more like that of unwoundedskin than control wounds (Figure 2). Both these changesreached statistical significance when scored using theblinded histological visual analogue scoring scale (Figure 3),leading us to conclude that this is a suitable model in whichto assess the effects of the treatments on wound reinner-vation. The histological visual analogue scale has beenpreviously described.24

A comparison of the innervation pattern and densitybetween the periphery and the centre of wounds revealedno difference between the two sites of measurement forany of the time points analysed in any of the treatmentgroups or controls. Because of this, statistical analysisbetween the different time points was carried out on thecombined data of peripheral and central wounds of allnerve fibre types.

The innervation density of treated and untreatedwounds as indicated by PGP9.5 immunostaining (Figures

Figure 1 Graph to show mean� SEM (nZ 6) areas of controland treated excisional wounds in cm2 at different times postwounding. The area of control wounds at day 5 are significantlysmaller (*p< 0.05) than the original wounds, and subsequentwounds are again significantly smaller.

4e6) followed a similar pattern in each group (Figure 7).There was a hyperinnervation of the wound, at day 42 postwounding in each of the treatment groups, although thispeak was not observed in the control wounds. Maximuminnervation density at day 42 was significantly greater inthe wounds treated with TGFb1 than the control group(p< 0.05). Overall nerve fibre densities in control andtreatment groups as indicated by PGP9.5 immunostainingreturned to baseline levels by day 84 (a mature scar).

CGRP immunoreactivity was decreased at day 7 postwounding, but then increased (Figure 6). A small peak of

Figure 3 Graph to show mean and SEM (nZ 6) scores fromthe visual analogue scale for assessment of scarring severity (orcollagen organisation) in 84-day-old control and treated scars.A significant improvement in scarring is seen with TGFb3, anda significant worsening with TGFb1 (*p< 0.05).

Figure 4 Collage showing positive and negative controls, andsections through untreated (control) wounds immunostainedfor PGP9.5 at 7, 42 and 84 days after wounding. Scale bar is200 mm.

Figure 5 Collage showing positive and negative controls, andsections through wounds treated with TGFb1 that have beenimmunostained for PGP9.5 at 7, 42 and 84 days after wounding.Scale bar is 200 mm.

Figure 6 Collage showing positive and negative controls, andsections through wounds treated with TGFb3 that have beenimmunostained for PGP9.5 at 7, 42 and 84 days after wounding.Scale bar is 200 mm.

The reinnervation pattern of wounds and scars after treatment e83

innervation density was seen at 14 days in control andtreatment groups (p< 0.05 vs 7 days for control, TGFb1 andTGFb3 treated wounds) (Figure 8), which then normalisedat 21 days. A statistically significant increase was seen at 42days (p< 0.05 vs 7 days for control and TGFb1 treatedwounds), but the innervation levels then returned to

Figure 7 Graph showing mean and SEM (nZ 6) of PGP9.5reinnervation of control wounds, and those treated to alter thescarring process. There was a significantly greater density ofnerve fibre immunoreactivity at day 42 between both treat-ment groups and the control wounds (*p< 0.05).

Figure 8 Graph showing mean and SEM (nZ 6) of CGRPreinnervation of control and treated wounds. Values at day 14were significantly greater than those found at day 7 and day 21wounds (*p< 0.05) with the exception that control woundCGRP densities were not significantly different between days14 and 21. Values for all groups at day 42 were significantlygreater (*p< 0.05) than those of unwounded skin, and day 7and day 21 wounds. There were no significant differencesbetween control wounds or either of the treatment groups atany time point.

e84 J. Henderson et al.

approximately those of unwounded skin by 84 days in allgroups.

SP immunoreactive nerve fibre densities were greatlydecreased at 7 days from unwounded skin in all treatmentgroups but then they increased steadily over the first 42days after wounding, reaching a density more than twicethat of unwounded skin. This finding was similar in alltreatment groups (Figure 9). This increase of SP nervedensity remained once the scar was mature at 84 days, andwas significantly greater than SP immunoreactivity densityin unwounded skin for controls and for each treatmentgroup (p< 0.05).

Figure 9 Graph showing mean and SEM (nZ 6) of SP rein-nervation of control wounds. SP immunostaining densities in allgroups at day 84 were significantly greater than in unwoundedskin. There were no significant differences in nerve fibreimmunoreactivity between control wounds or either of thetreatment groups at any time point.

Discussion

The excisional wound model used in this study was chosenbecause it produces a scar large enough to allow compar-ison of reinnervation at both the edges and centre of thewound. The defect heals by granulation, and althoughthere is a large degree of wound contraction, a thickcollagenous scar is produced. We did not assess scarcontraction at early enough points to evaluate fullywhether it was affected by the treatments. Treatment withTGFb1 has previously been found to accelerate incisionalwound contraction and increase wound strength aftera single application.25,26 Because the scar-altering treat-ments changed the degree of collagen organisation in thescars significantly, it is reasonable to assume that this isa suitable model in which to assess the effects of thetreatments on wound reinnervation.

TGFb isoforms are structurally similar to nerve growthfactor and increase NGF levels in rat neonatal brain17 andperipheral nerve18 injury. NGF-like action of TGFb isoforms,or a secondary increase in NGF production might accountfor the finding that both TGFb1 and TGFb3 which haveopposite effects on the collagen matrix of the scar bothincreased the maximum innervation density. Treatmentsthat decrease scarring might facilitate nerve fibre growthand pattern through the matrix simply by a templatingeffect.

Substance P increases the actions of metalloproteinasesleading to collagen breakdown.27 It is conceivable that SPmay act as a facilitator of the passage of nerves or bloodvessels through the collagen matrix; SP containing nervefibres might forge their own path through scars by locallyincreasing collagen breakdown.

In other wound models that there is an initial hyper-reinnervation, and the overall nerve density then recedesto approximately that of uninjured controls.28e30 This wasnot the pattern that we found with control wounds, asoverall innervation did not reach densities greater thanunwounded skin. These results from other groups, however,correspond to our findings of the reinnervation pattern ofCGRP immunopositive nerve in all wounds, and PGP9.5 intreated wounds. Sensory nerve hyperinnervation was foundin neonatal foot skin wounds up to 84 days, comprising bothC and Ad nerve fibres.31 We found this pattern only with SPimmunopositive C fibres. Such differences between ourfindings and those of others might be explained by differ-ences in the wound model. In experiments of this type inmice it is assumed that a scar at 84 days is mature. It wouldbe interesting to see if the density of SP positive nervefibres returns to control levels at a later time (e.g. 150days).

The proportion of wound and scar reinnervation thatconsists of non-SP non-CGRP nerve fibres is also greatlyreduced in all wounds. If treatments that reduce scarringlead to an increase in the proportion of nerve fibres that areSP and CGRP immunopositive, we might worry that treatedscars might have more severe or more frequent symptomsof pain or pruritis. However, the density of such fibres wasnot significantly increased from controls (Figures 5 and 6)and in clinical trials of TGFb3 on humans, no pruritis or painfrom the scars has been experienced.16 It is also clear that

The reinnervation pattern of wounds and scars after treatment e85

SP and CGRP levels are greatly increased in all wounds, butthat not all scars are pruritic or painful.

Previous studies finding high levels of SP or CGRP inscars have been associated with the scar being hypertro-phic.9,10 This study provides evidence that SP levelsremain elevated in normotrophic scars, and those in whichthe scarring process has been reduced, whilst CGRP levelsrise to greater than unwounded skin during the healingprocess, but then return to similar levels in the scar. Micerarely produce hypertrophic scars; it seems likely thatelevated levels of SP are not directly responsible for scarsbeing hypertrophic. Increased SP density in hypertrophicscars may represent a response to the hypertrophy ratherthan the cause, representing part of the remodellingprocess.

This is the first investigation into the effects on woundreinnervation of scar-altering treatments. Although somechanges to the reinnervation pattern were produced by thetreatments, the overall pattern of wound reinnervationfollowed a similar pattern to control wounds, with theexception that both treatments caused an increase in nervedensity at day 42. No long-term changes in the reinnerva-tion pattern (day 84) were caused by either treatment.These findings are reassuring as TGFb3 is in clinical trials asan antiscarring agent, although phase III clinical trials failedto reach the desired commercial endpoint. TGFb3 has beenfound to significantly improve scar appearance, reduce scararea, and normalise collagen fibre arrangement within thescar.32,33,34 Further work in humans may include clinicaland immunohistological evaluation of scars to assesssensation and nerve anatomy in control, hypertrophic andtreated scars.

The increase of innervation density at day 42 could bedirectly caused by actions of the treatments, or otherwiseby a reaction to changes in the collagen matrix. Although itdid not reach statistical significance, day 42 is also the timepoint at which control wounds were maximally innervated.Nerve growth factor, CGRP and SP are all mediators ofinflammation and healing, whilst injured tissues releaseNGF. Wounded tissues stimulate reinnervation, whilstneurotransmitters stimulate the healing process.35 Theinteractions are very complex, and it is unclear why bothTGF beta isoforms caused a significant increase in inner-vation density over control wounds at that time point, butan alteration in the collagen matrix may be the most likelyexplanation.

Interleukin 10, another putative antiscarring treatmenthas been found to cause transient elevation of CGRPimmunoreactive nerve fibres during the healing process,which was associated with transient hypervascularisation.36

Negative pressure wound therapy accelerates wound heal-ing, and is associated with increased expression of SP,CGRP, and NGF along with increased nerve fibre density inthe adjacent dermis.37 It might be interesting to correlatechanges in the innervation pattern of wounds with directmeasurements of neurotrophins and nerve growth factor, aswell as relevant gene expression profiles, and to investigatethe effects of scar-altering treatments on such factors.

The molecular mechanisms signalling healing, nerve andblood vessel growth appear to be linked, but it is not yetknown what stimulates the differential growth of thedifferent subtypes of nerve fibres seen in this study.

Funding

The study was supported by funds from Renovo Ltd and TheRoyal College of Surgeons of Edinburgh.

Conflict of interest

JH was funded by Renovo, of which MWJF is co-founder andCEO. Renovo is conducting clinical trials of TGFb3 (Juvista/Avotermin) as an antiscarring agent.

Experimental animals

All experiments were carried out in accordance with the UKanimal act (1986) and with appropriate legal and ethicalapproval.

Acknowledgements

The authors are grateful to Bill Bennett, Mary Birch, HayleyWillis and Cristina Mantovani for practical help and adviceand to Hugh Laverty at Renovo Ltd for the gift of TGFb3 andbuffer. The study was generously supported by funds fromRenovo Ltd and The Royal College of Surgeons of Edinburgh.

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